Real-Time
complex Size
monitoring for Transfection

Critical Challenges in Transfection Complex Size Monitoring

In upstream processing for gene therapy products such as AAVs and Lentiviral Vectors, transient transfection is a commonly used approach.

The step before cells are transfected

A critical – but often under-monitored – step happens before any cells are transfected: the complexation reaction, where plasmid DNA (pDNA) or RNA binds to a transfection reagent (e.g., PEI or cationic lipids) to form transfection complexes (polyplexes or lipoplexes) during mixing and incubation.
The NanoLabSizer measures the size of these complexes as they form and grow – not the transfection of cells.

Complex size can change while the complexes form and grow, and that size strongly influences downstream transfection performance.

Complex formation affects performance

When the optimal complex size window is narrow, small shifts during mixing, resting time, or transfer can lead to variability, repeat work, and yield loss – yet size drift during complex formation often goes unnoticed.

That’s why monitoring complex size during complexation – in real time and without sampling – can make transfection outcomes more consistent.

Transfection complexes include:
Polyplexes (polymer–nucleic acid nanoparticles) and lipoplexes (lipid–nucleic acid nanoparticles).
In AAV manufacturing, these non-viral vectors are used to deliver plasmid DNA (pDNA) to host cells (e.g., HEK293) for recombinant AAV production.

Top 5 challenges

1. Maintaining sterility when sampling
2. Avoiding destructive measurements
3. Getting real-time insight during complexation
4. Reducing variability from handling and delayed feedback
5. Standardizing and transferring complexation protocols

The NanoLabSizer, launched in 2026, enables real-time, non-invasive nanoparticle size analysis using Spatially Resolved Dynamic Light Scattering (SR-DLS).

• No need to stop your process
• No sample extraction
• No compromise on sterility

Measure directly through any transparent container, including:

• IV bags
• Glass or plastic vials
• Prefilled syringes
• Bioreactor bags

Understanding

Helps to accelerate development, scale-up and validation

• Direct link between complex size and batch yield
• Continuous, data-rich size–growth curves instead of current offline approach
• More representative size data by avoiding sampling and measurement delays

Efficiency

Shorter time, less experiments, less manhours

• Eliminates destructive sampling and “sacrifice” transfection batches
• Continuous kinetics reduces the number of experiments
• No skilled lab personnel or sample handling needed

Direct savings

Costs of materials, waste, number of batches, labour

• No extra non-sterile batch for offline size analysis
• 15–30% higher titre/yield from more accurate, scalable recipes
• No sampling disposables or hands-on time thanks to continuous readout

The NanoLabSizer Measures

• Complex size and growth, before cell transfection
• Data collection every 5-10 seconds
• Possible Aggregation
• Stabilization points

Processes you can follow during complex formation:

• mixing strategy,
• complexation/incubation time,
• transfer/hold time,
• formulation settings (e.g., N/P ratio, buffer, concentration).

Optimizing the size of pDNA-PEI complexes is critical because both overly small and overly large particles undermine transfection performance.
The same principle applies to lipoplexes and lipopolyplexes, where size and aggregation during complexation can shift performance.

• How can you measure particle size CQAS in (sterile) packaging?
• Whitepaper – How do results from different DLS instruments compare?
• Whitepaper – Inline detection of aggregates and oversized particles